Recent advances in gene technology and molecular biology have made diagnoses of diseases such as infectious diseases and genetic diseases possible at levels of DNA and RNA. Gene analysis has been immensely facilitated by the method for the nucleic acid amplification such as polymerase chain reaction (PCR; Science, 230: 1350-1354,1985), NASBA (Nucleic Acid Sequence Based Amplifications, Nature, 350, 91-92,1991;
Japanese Patent No. 2648802; Japanese Patent No. 2650159) and LAMP (Loop mediated isothermal amplification of DNA, Japanese Patent Laid-open Publication No. 2001-242169) in particular, through which a detection of a trace amount of nucleic acids in biological samples, which had been extremely difficult to detect, has become possible.
PCR is a method which enables to amplify an objective DNA fragment hundreds of thousands-fold by repeating a cycle of a dissociation of double strands of DNA into single strands, binding of primers to sequences flanking specific region of DNA strands and a DNA synthesis by a DNA polymerase. The method was invented by Mullis and others, and is described in Japanese Patent Laid-open Publication No. S61-274697. PCR can be used as a sensitive method for analyzing nucleic acids from various samples and particularly useful for analyzing nucleic acids in samples derived from body fluids of animals. Accordingly, PCR is utilized for diagnoses of infectious diseases, genetic diseases and cancers. Moreover, the method is suitable for a typing of DNA upon a transplantation and an identification of a parent-child relationship. In these cases, peripheral blood is often used as a sample to be examined.
One of drawbacks of PCR is that the reaction is interfered by pigments, proteins, sugars and other unknown contaminants. In fact, it is widely known that in addition to the most frequently used a thermoresistant DNA polymerase, a TaqDNA polymerase derived from Thermas aquaticus, a number of DNA polymerases are strongly inhibited by the presence of a trace of contaminants derived from body fluids.
RT-PCR is a method where RNA extracted from, for instance, tumor tissues, is converted to cDNAs by a reverse transcriptase (RT), by using an origo (dT) or a random hexamer as a primer, part of cDNAs are utilized for a PCR amplification, then used for detection. A case using this method to diagnose a fibloblastoma is reported (Hokkaido Igaku Zasshi, p.135-141, Vol. 66(2), 1991). Since RT-PCR relies on mRNA which is prone to be degraded, a quick treatment is required after collection of a sample. Furthermore, special care has to be taken to prevent the sample from a contamination of others, as RT-PCR is an extremely sensitive method for detection of DNA.
LAMP is a method for a gene amplification by using more than two sets of primers comprising special primers that form hairpin structures at a distal ends of amplification products as strand displacement reaction proceeds. Amplification products consisted of many repeated structures, whose unit of repeating structure consists of a complementary region in the same strands wherein base sequences of a double-stranded nucleic acid are reversed. Although amplification products of LAMP can be detected by a publicly known method for detection of double-stranded DNA, a detection method taking advantage of their unique structure is reported (Japanese Patent Laid-open Publication No. 2001-242169, WO 00/28082).
Other lines of the method for the gene amplification, TMA, NASBA, and 3SR, are characterized in the use of primers carrying the T7 promoter sequence in one of two primer strands. Therefore, when these primers are hybridized to a target DNA or RNA followed by a reverse transcription (a DNA synthesis using RNA or DNA as a template), a double-stranded DNA with a T7 promoter is synthesized. A large amount of RNA are synthesized as the amplified product by addition of a T7 polymerase to this reaction system, as a result of the activation of the T7 promoter followed by expression of downstream genes. In TMA, NASBA and 3SR, amplification cycles described above proceed at a constant temperature.
A system for detection of nucleic acid utilizing means for nucleic acid amplification is generally composed of a process for treatment of biological materials, a process of extracting of nucleic acids, a process for dispensing measuring reagents, a process for the nucleic acid amplification and a process for an assessment of amplification products. A process of preliminary treatment for the extraction of nucleic acids is needed prior to the nucleic acid amplification by methods such as a PCR method. The methods for nucleic acids extraction have been used wherein gene-containing materials are degraded by an enzyme,a surfactant or a chaotropic agent, then a treatment by phenol (Biochemica et Biophysica Acta, 72: 619-629,1963), alkaline conditions (Nucleic Acid Research, 7: 1513-1523,1979), or phenol-chloroform and so on are carried out. Recently, ion-exchange resins, a glass filter, glass beads or a reagent having an aggregating activity for proteins are used in the process of nucleic acids extraction.
As a system utilizing a nucleic acid-binding carrier for an extraction of nucleic acids, a method of using glass particles and sodium iodide (Proc.Natl.Acad.Sci.USA, 76-2: 615-619, 1979) and a method using hydroxyapatite (Japanese Patent Laid-open Publication No; S63-263093) and so on are reported. In these methods, whereas a use of toxic reagents such as an organic solvent is limited, instead there is a problem in that it takes long time to isolate nucleic acids due to repeated centrifugation during the process, making it difficult to handle many samples at a time.
As described, conventional methods for an isolation of nucleic acids have disadvantages in that they employ dangerous reagents such as the organic solvent and alkaline substances, they require centrifugation steps, making it difficult to handle many samples at a time. The disadvantage becomes even more serious in view of automation, in which many samples are treated with high reproducibility to reduce personal costs.
As a method for automating the process for the extraction of nucleic acids, there is a method of using nucleic acid-binding silicate particles and chaotropic ions (J. Clinical Microbiology, 28-3: p.495-503, 1990, Japanese Patent Publication No. 2680462). In this method, by mixing nucleic acid-binding silicate particles and chaotropic ion having an ability to isolate nucleic acids in the sample and the sample, nucleic acids are first combined with nucleic acid-binding silicate particles, allowing a separation of a solid phase from a liquid phase to remove contaminants in the sample, followed by an elution of nucleic acids bound to nucleic-acid binding silicate particles.
Acid treatment of biological samples comprising nucleic acids is reported (International Publication WO 01/00813) as means for removing inhibitory substances on the nucleic acid amplification. However, in spite of the description on the possibility of acid treating biological samples at any given stage for the extraction of nucleic acids from the biological samples, it remains difficult to carry out rapid nucleic acid amplification since no description has been given with regard to conducting the nucleic acid amplification without isolation and the purification of nucleic acids.
A method is reported wherein samples are brought into contact with an anionic solid phase to facilitate the binding of inhibitory substances on the nucleic acid amplification, as means for reducing the inhibition on reaction of nucleic acid amplification (Japanese Patent Laid-open Publication No. 1997-173065). However, it is still difficult to conduct a rapid nucleic acid amplification, since no description was given herein, with regard to carrying out the nucleic acid amplification without isolation and purification of nucleic acids.
The above-mentioned methods for the nucleic acid amplification have been put to practical use in the field of diagnoses of tumors and cancers, a detection of virals as well as bacterial infections, and a detection of various genetic diseases (Handbook of the method of clinical examination, 31st edition, Kanahara & Co., Ltd, published on Sep. 20th, 1998).
However, many aspects are left to be improved in terms of a necessary time for a measurement and sensitivity in the detection of nucleic acids by way of the nucleic acid amplification.